Project Summary The goals of this renewal are to understand the mechanism of promoter proximal pausing and to understand the function of the CTD of RNA polymerase II (Pol II) in transcription. Promoter proximal pausing by Pol II is widespread in Chordates and Drosophila and is now recognized as a ubiquitous step in the transcription cycle of metazoans. Regulation of the duration of the pause significantly impacts levels of gene expression. Pausing requires DSIF and NELF but the mechanism by which these proteins cause pausing is not known. In addition, the CTD of Pol II is widely thought to somehow regulate pause release. The CTD is also involved with transcription initiation, elongation and termination and with RNA processing and chromatin structure modulation. Using Drosophila as a model system, the roles of the following in pausing will be investigated: 1) nucleic acid interacting domains of the Spt5 subunit of DSIF, 2) the RNA recognition motif in the NELF-E subunit of NELF, 3) the CTD and linker regions in the Rpb1 subunit of Pol II. To identify additional proteins involved in pausing, a recently developed BioTAP-XL method that combines crosslinking, tandem affinity purification and mass spectrometry will be used to determine the protein composition of native paused complexes isolated from Drosophila embryos. Investigation into the function of the CTD stems from our recent finding that the sequence composition of the CTD can be markedly altered without disrupting its capacity to support Drosophila development and proliferation. In addition, fusing the CTD to green fluorescent protein (GFP) causes GFP to associate with transcriptionally active regions in Drosophila polytene chromosomes. These results prompt this hypothesis: the CTD of Pol II functions as a signal sequence that causes Pol II to partition into transcription compartments. To test this hypothesis, various CTD mutants will be evaluated to determine if their ability to support Pol II function correlates with their ability to cause GFP to partition into transcription compartments. The effects of CTD mutants on Pol II interactions with the hsp70 heat shock gene will also be compared to the targeting activity of the CTD to elucidate whether this targeting activity can be linked to specific steps in the transcription cycle. There is growing interest in the possibility that transcription occurs in liquid phase separated compartments and FUS is a candidate protein that could be involved in causing phase separation. Translocations involving FUS have been linked to liposarcomas and mutations in FUS have been linked to motor neuron disease, amyotrophic lateral sclerosis. To investigate the function of FUS in transcription, FUS and a functionally related protein, TDP-43, will be depleted from Drosophila salivary glands and the effects on CTD function will be analyzed. Since normal human FUS but not pathological mutants have been shown to complement FUS mutations in Drosophila, the impact of these human FUS derivatives on CTD function in Drosophila will be evaluated. The combination of biochemical, cytological, and genetic approaches makes this project uniquely suited to achieve its goals.